15 research outputs found
Lymphatic Capillaries in Aging
The lymphatic system is responsible for fluid drainage from almost every organ in the body. It sustains tissue homeostasis and is also a central part of the immune system. With the discovery of cell-specific markers and transgenic mouse models, it has become possible to gain some insight into the developmental and functional roles of lymphatic endothelial cells (LECs). Only recently, a more direct regulatory role has been assigned to LECs in their functions in immunity responses and chronic diseases. Here, we discuss the changes occurring in aged lymphatic system and the role of lymphatic capillaries in some age-related diseases and experimental animal models.(c) 2020 The Author(s) Published by S. Karger AG, Base
Elevated sodium leads to the increased expression of HSP60 and induces apoptosis in HUVECs
<div><p>Atherosclerosis is the leading cause of death in the world. We have previously shown that expression of heat shock protein 60 (HSP60) on the surface of endothelial cells is the main cause of initiating the disease as it acts as a T cell auto-antigen and can be triggered by classical atherosclerosis risk factors, such as infection (e.g. <i>Chlamydia pneumoniae</i>), chemical stress (smoking, oxygen radicals, drugs), physical insult (heat, shear blood flow) and inflammation (inflammatory cytokines, lipopolysaccharide, oxidized low density lipoprotein, advanced glycation end products). In the present study, we show that increasing levels of sodium chloride can also induce an increase in intracellular and surface expression of HSP60 protein in human umbilical vein endothelial cells. In addition, we found that elevated sodium induces apoptosis.</p></div
Surface staining of HSP60 on HUVECs treated with increasing sodium concentrations.
<p>(A) Representative images of nuclei (Hoechst), β-actin (A488) and HSP60 (A568) on HUVECs fixed with 2% PFA + MetOH or with 1% PFA. Scale bar = 50 μm. (B) MFI of surface HSP60 expression out of CD31<sup>+</sup> endothelial cells. MFI of three donors ± SEM is shown. One-way ANOVA analysis was performed, p-value = 0.3916. (C) Representative images of surface staining of HSP60 under each experimental condition. Scale bar = 50 μm. (D) Quantification of surface HSP60 fluorescence staining intensity as explained in the Material and Methods. Mean CTCF values ± SEM combined from two independent experiments. Each dot represents the CTCF readout from one donor (n = 6). (E) Linear regression of values presented in (D). Dotted lines show the 95% confidence interval. Mean ± SD. CTCF = corrected total cell fluorescence. mM = millimolar. MFI = median fluorescence intensity.</p
Intracellular HSP60 expression in HUVECs treated with increasing sodium concentrations.
<p>(A) Representative images of nuclei (Hoechst) and HSP60 (A568) staining under each experimental condition, on HUVECs fixed with 2% PFA + MetOH. Scale bar = 20 μm. (B) Quantification of HSP60 fluorescence staining intensity as explained in Material and Methods. Mean CTCF values ± SEM combined from two independent experiments. Each dot represents the CTCF readout from one donor (n = 6). (C) Linear regression of values presented in (B). Dotted lines show 95% confidence intervals. Mean ± SD. *<i>p</i><0.05, **<i>p</i><0.01. CTCF = corrected total cell fluorescence. mM = millimolar.</p
Correlation between HSP60 expression and apoptosis in HUVECS.
<p>Pearson correlation analysis. shows a significant correlation of intracellular and surface expression of HSP60 with the number of apoptotic cells, as determined by flow cytometry. Each dot represents the mean CTCF and corresponding percentage of apoptosis (Annexin V<sup>+</sup>) of 6 samples. CTCF = corrected total cell fluorescence.</p
Quantification of total cells and apoptotic cells in HUVECs treated with increasing sodium concentration.
<p>(A) Representative images of HUVECs treated with 137 mM and 188 mM sodium, fixed with 2% PFA + MetOH and stained with Hoechst and anti-β-actin A488, and their subsequent enumeration using FIJI, as explained in Material and Methods. The number of cells per image is displayed in red under particle analysis. Scale bar = 100 μm. (B) Scatter plot of enumeration from acquired images of HUVECs treated with increasing sodium concentrations. Each dot represents number of cells per image. Mean ± SEM (C) Linear regression of values shown in (B). Mean ± SD (D) Representative flow cytometric gating strategies for enumeration of cells and Annexin V<sup>+</sup> cells (apoptotic cells). Linear regression of FACS results for number of cells and percentage of apoptotic cells. Mean ± SD (E) Summary of flow cytometric enumeration of live and apoptotic cells (Annexin V<sup>+</sup>). The percentage of apoptotic cells within the total number of cells is shown on top of the bars. Data are from one experiment (n = 3). ***<i>p</i><0.001. CTCF = corrected total cell fluorescence. mM = millimolar. B&W = black and white. No. = number.</p
A role for the nuclear receptor NR2F6 in peritoneal B cell homeostasis
B cells are key mediators of humoral immunity. Mature B cells fall into various sub-classes that can be separated by their ontogeny, expression of cell surface markers, anatomical location, and function. B1 subsets play important roles in natural immunity and constitute the majority of B cells in newborns. In the adult, B1 cells predominate in the pleural and peritoneal cavities, while the mature B2 follicular subset makes up the major fraction of B cells in lymphoid tissue, although important subsets of antibody-secreting B1 cells are also present at these sites. B1 cells are the main producers of natural IgM but can also contribute to elimination of some pathogens, while B2 cells primarily mediate response to foreign antigens. The differential molecular underpinning of the B1 and B2 subsets remains incompletely understood. Here we demonstrate that germline-deficiency of the orphan nuclear receptor NR2F6 causes a partial loss of B1b and B2 B cells in the peritoneum while leaving peritoneal B1a cells unaltered. A competitive bone marrow chimera in Nr2f6(+/+) host mice produced similar numbers of Nr2f6(+/+) and Nr2f6(-/-) peritoneal B1b and B2 cells. The proliferation of Nr2f6(-/-) peritoneal B cells was not altered, while the migration marker CXCR5 was reduced on all subsets but Beta7-integrin was reduced only on peritoneal B1b and B2 cells. Similarly, B1b and B2 but not B1a cells, exhibited significantly reduced survival
Immune-interacting lymphatic endothelial subtype at capillary terminals drives lymphatic malformation
Oncogenic mutations in PIK3CA, encoding p110α-PI3K, are a common cause of venous and lymphatic malformations. Vessel type-specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit prolymphangiogenic macrophages to promote progressive lymphatic overgrowth. Mouse model of Pik3ca-driven vascular malformations showed that proliferation was induced in both venous and lymphatic ECs but sustained selectively in LECs of advanced lesions. Single-cell transcriptomics identified the iLEC population, residing at lymphatic capillary terminals of normal vasculature, that was expanded in Pik3ca mice. Expression of pro-inflammatory genes, including monocyte/ macrophage chemokine Ccl2, inPik3ca-iLECs was associated with recruitment of VEGF-C-producing macrophages. Macrophage depletion, CCL2 blockade, or anti-inflammatory COX-2 inhibition limited Pik3ca-driven lymphangiogenesis. Thus, targeting the paracrine crosstalk involving iLECs and macrophages provides a new therapeutic opportunity for lymphatic malformations. Identification of iLECs further indicates that peripheral lymphatic vessels not only respond to but also actively orchestrate inflammatory processes
Loss of the orphan nuclear receptor NR2F6 enhances CD8(+) T-cell memory via IFN-gamma
Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127(-)KLRG1(+)) or memory precursors cells (MPECs, CD127(+)KLRG1(-)) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8(+) memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-gamma, TNF alpha, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6(-/-) OT-I T-cells showed that the augmented memory formation is CD8(+) T-cell intrinsic. Although the relative difference between the Nr2f6(+/+) and Nr2f6(-/-) OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-gamma responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8(+) T-cell responses. Following a secondary adoptive transfer into naive congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-gamma mediated. IFN-gamma blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8(+) T cells may have therapeutic potential for enhancing early IFN-gamma production and consequently the functionality of memory CD8(+) T cells in vivo
Analisis Pohon Berstruktur Menggunakan Metode CHAID pada Data Respons Ordinal
Eksplorasi data merupakan suatu hal yang penting dilakukan sebelum menganalisa data dengan metode lain. Salah satu metode eksplorasi untuk data respons kategorik adalah metode CHAID (Chi-square Automatic Interaction Detection). Tulisan ini berisi tentang kajian teori metode CHAID dan penerapannya yaitu menganalisis faktor-faktor yang berpengaruh pada peningkatan omset USAha anggota koperasi simpan pinjam (KSP) sebagai respons ordinal. Hasil kajian menunjukkan bahwa metode CHAID tidak hanya dapat digunakan sebagai metode eksplorasi tetapi juga mampu menggunakan struktur hubungan antara variabel respons kategorik dengan serangkaian variabel penjelas serta interaksi antar variabel penjelas. Struktur hubungan ini digambarkan dengan suatu pohon berstruktur